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Immunoglobulin M antibodies against CRA and FRA recombinant antigens of Trypanosoma cruzi in chronic chagasic patients
Human Immunology 72 (2011) 402– 405
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Immunoglobulin M antibodies against CRA and FRA recombinant antigens of Trypanosoma cruzi in chronic chagasic patients Romero H.T. Vasconcelos a, Elisa A.N. Azevedo a, Maria G.A.M. Cavalcanti b, Edimilson D. Silva c,d, Antonio G.P. Ferreira c,d, Clarice N.L. Morais a, Yara M. Gomes a,d,* a
Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhâes/Fiocruz, Recife, Brazil Hospital Universitàrio Oswaldo Cruz, Universidade de Pernambuco, Recife, Brazil c Departamento de Reativos para Diagnòstico, Bio-Manguinhos/Fiocruz, Rio de Janeiro, Brazil d Programa Integrado de Doenåa de Chagas/Fiocruz, Rio de Janeiro, Brazil b
A R T I C L E
I N F O
Article history: Received 29 October 2010 Accepted 22 February 2011 Available online 1 March 2011
Keywords: Immunoglobulin M Recombinant antigens Trypanosoma cruzi Chagas disease
A B S T R A C T
Previous works of our research group have demonstrated aspects of the humoral immune response of chronic Chagas disease using the cytoplasmatic repetitive antigen (CRA) and the flagellar repetitive antigen (FRA) of Trypanosoma cruzi. The aim of this work was to analyze the presence of specific immunoglobulin M (IgM) antibodies in chronic chagasic patients using these recombinant antigens of T. cruzi. The positivity of IgM in chronic chagasic patients against CRA and FRA antigens was determined by indirect enzyme-linked immunosorbent assay. We reported no statistical significant differences between the levels of IgM for both recombinant antigens and the different chronic clinical forms of Chagas disease. However, a small proportion of chronic chagasic patients analyzed in this study was positive for this antibody isotype. The findings of this study indicate that the IgM antibodies cannot be used to elucidate the differences in the profile of humoral immune response among chronic chagasic patients with different clinical forms using the CRA and FRA recombinant antigens of T. cruzi. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Chagas disease is a parasitic infection caused by the protozoan Trypanosoma cruzi. In the acute phase of the disease, usually developed with nonspecific symptoms, the blood parasitemia and the presence of immunoglobulin M (IgM) antibodies against T. cruzi are diagnostic criteria to confirm the infection [1]. Acute Chagas disease is followed by a chronic asymptomatic phase, also called indeterminate (IND) form. In this chronic form the patient enters a period of latency with no evidence of infection, other than positive serology for IgG against T. cruzi. About 20 to 30% of chronically infected individuals may develop severe forms with cardiac (CARD) and digestive (DIG) manifestations or both (CD) after 10 to 20 years of the initial infection [2]. The pathology of the lesions that lead to severe forms remains unknown [2,3]. However, some authors recognized that the human immune response to parasite antigens is related to the different clinical forms of the disease [3–5]. Magnani et al. [6] demonstrated that chronic chagasic patients have increased levels of IgM, probably because of antigenic variability of T. cruzi. Morgan et al. [7] demonstrated that chronic symptomatic patients present high levels of IgM against T. cruzi. According to Morgan et al. [7], this class of
* Corresponding author. E-mail address: yara@cpqam.fiocruz.br (Y.M. Gomes).
antibodies is first produced against T. cruzi antigens, but maintenance of this isotype in tissues and serum is directly associated with cross-reactivity among T. cruzi and host antigens. Although some studies have demonstrated the presence of IgM antibodies in the chronic phase of Chagas disease, only a few were conducted using pure and chemically defined antigens [8 –10]. BetÕnico et al. [11] compared the IgM positivity in chronic Chagas disease of a synthetic tripeptide with an alkaline extract of T. cruzi. The synthetic peptide exhibited a significantly lower number of positive samples when compared with the alkaline extract. In the present study we used two recombinant antigens of T. cruzi, chemically characterized and with a repetitive epitope structure, to evaluate the levels of IgM in chronic chagasic patients with different clinical forms. The cytoplasmatic repetitive antigen (CRA) has a repeated sequence of 14 amino acids and is expressed in epimastigote and amastigote forms of T. cruzi, whereas the flagellar repetitive antigen (FRA), present in the epimastigote and trypomastigote forms, has a repeated sequence of 68 amino acids [12,13]. VerÈosa et al. [14] studied the IgG isotypes against the CRA and FRA antigens in chronic chagasic patients and demonstrated that the IgG2 isotype against the FRA antigen was able to distinguish patients with the CARD form from those with the IND form. Recently, Vasconcelos et al. [15] studied the serum levels of IgA antibodies against these recombinant antigens and demonstrated that
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.02.015
R.H.T. Vasconcelos et al. / Human Immunology 72 (2011) 402– 405
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the digestive forms of the disease present high levels of this isotype of immunoglobulin. Given the importance of the human immune response against the parasite antigens in the development and maintenance of chronic clinical forms of Chagas disease, the aim of the present study was to evaluate the levels of specific IgM against the CRA and FRA recombinant antigens of T. cruzi in the serum of chronic chagasic patients. It is possible that this isotype is involved in the pathology of severe clinical forms of the disease. 2. Subjects and methods 2.1. Study population Serum samples were collected from 96 chronic chagasic patients selected at the Oswaldo Cruz University Hospital of the State University of Pernambuco, Brazil. The selection of individuals was based on the following criteria: (1) clinical tests for characterization of the clinical forms as established by the World Health Organization [1]; (2) two positive serologic tests for Chagas disease; and (3) not having undergone etiologic treatment. According to their clinical status, the patients were classified as having IND (n ⫽ 24), CARD (n ⫽ 25), DIG (n ⫽ 20), or CD (n ⫽ 27) forms of the disease. To establish a cutoff point, serum was also collected from 14 non-chagasic individuals. Negative serologic results for infection by T. cruzi in two tests and never having received a blood transfusion were the inclusion criteria for this group.
Fig. 1. Reactivity indexes of immunoglobulin M against CRA antigen in chronic chagasic patients. Symbols represent the reactivity indexes, calculated as the mean OD of each sample divided by the cutoff of their plates. The horizontal bars represent the mean of reactivity indexes for each group of patients studied. IND, indeterminate form; CARD, cardiac form; DIG, digestive form; CD, cardiodigestive form; CRA, cytoplasmatic repetitive antigen; OD, optical density; CO, cutoff.
2.2. Ethical considerations All individuals involved in this study participated on a voluntary basis and signed the Terms of Free and Informed Consent. The methods of inclusion and the protocols used were approved by the Ethics Committee of the Aggeu MagalhÄes Research Center (Number 070/2008). 2.3. Recombinant antigens The CRA and FRA antigens of T. cruzi were prepared at the Bio-Manguinhos Department of Diagnostic Reactions and were obtained as previously described by Krieger et al. [13]. 2.4. Detection of IgM antibodies against CRA or FRA antigens Enzyme-linked immunosorbent assay (ELISA) plates (Nalge Nunc International Corporation, Rochester, NY) were coated with 100 L/well of CRA or FRA recombinant antigens at previously established concentrations (0.625 g/mL) diluted in a 0.05 mol/L carbonate– bicarbonate buffer, pH 9.6. After being blocked with phosphate-buffered saline containing 0.05% Tween 20 (PBST) and 1% bovine serum albumin, the plates were incubated with serum diluted 1:100 in PBST and then with biotin-conjugated immunoglobulin (anti-IgM; Sigma, St. Louis, MO) diluted 1:4000 in PBST and 1% bovine serum albumin and subsequently with peroxidaseconjugated streptavidin (GE Healthcare, Buckinghamshire, UK) diluted 1:6000 in PBST. The immunocomplex was revealed by the addition of 0.01% o-phenylenediamine and 0.01% hydrogen peroxide diluted in a 0.077 mol/L citrate–phosphate buffer, pH 5.0. The reaction was terminated by adding 2.5 N sulfuric acid, and the absorbance was measured using an ELISA plate reader (Bio-Rad 3550, Bio-Rad Laboratories, Vienna, VA) at 490 nm. All samples were tested in duplicate. The cutoff point was established for each plate as the mean of optical density (OD) of non-chagasic individuals plus 2-fold standard deviation and the results were expressed as a reactivity index, as previously described by Antas et al. [16], whereby the value of the mean of the OD of each sample was divided by the cutoff value of their respective plates to enable comparison of the results from different plates. Reactivity indexes with values higher than 1.0 were considered positive [16].
2.5. Statistical analysis The R software package was used to analyze the results. Bartlett, analysis of variance, and Tukey tests were applied to evaluate the differences between the indexes calculated for the samples in each group of patients. Data are presented as means ⫾ standard error of the mean, and the significance level for all conclusions was set at 5%. 3. Results The mean levels of IgM antibodies against CRA or FRA recombinant antigens of T. cruzi were just low in almost all chronic chagasic patients studied. However, a small proportion of chronic chagasic patients had high levels of this isotype. The positivity of IgM antibodies in the serum of the chronic chagasic patients evaluated in this study was 10.42% for the CRA antigen and 11.46% for the FRA antigen. No significant statistical differences were observed in the serum levels of IgM when we compared the patients with different clinical forms by their reactivity indexes using the CRA antigen (Fig. 1). The FRA antigen antibody isotype was higher in patients with the CARD and CD forms when compared with patients with the IND and DIG forms, although these differences were not statistically significant (Fig. 2). 4. Discussion The immunologic basis for the variation of clinical manifestations among patients with chronic T. cruzi infection remains unclear [2]. Previous studies recognized that a particular antibody isotype may be related to chronic clinical manifestations of the disease [4 – 8]. This observation led us to examine a possible association between antibody isotype profile against CRA and FRA recombinant antigens of T. cruzi in chronic chagasic patients and different chronic clinical forms of the disease, with a view to using these antibodies as prognostic markers for the evolution of Chagas disease. In previous works of our research group, the IgG2 isotype against the FRA antigen was associated with the CARD form [14] and the IgA isotype against both antigens was associated with the
404
R.H.T. Vasconcelos et al. / Human Immunology 72 (2011) 402– 405
chronic phase of Chagas disease. The differences observed in the percentage of positive samples likely occurred because the antigens used in the studies do not share the same epitopes. These findings agree with the hypothesis that anti-T. cruzi IgM antibodies are only occasionally reported in chronic chagasic patients because of antigenic variability of the parasite [10]. According to Magnani et al. [6], the constant antigenic variability is a strategy of immune evasion used by T. cruzi and other protozoans to survive and reproduce inside the host. Moreover, the hypothesis that this isotype of antibodies are produced and maintained in the chronic phase of Chagas disease because of the cross-reactivity with host antigens [7] is not supported by these studies. If this hypothesis were correct, almost all chronic chagasic patients would present high levels of IgM because of producing an immune response against selfantigens. Taking the results of this study into account, the IgM antibodies against the CRA and FRA recombinant antigens of T. cruzi cannot be used to elucidate differences in the profile of humoral immune response among chronic chagasic patients with different clinical forms. Fig. 2. Reactivity indexes of IgM against FRA antigen in chronic chagasic patients. Symbols represent the reactivity indexes, calculated as the mean OD of each sample divided by the cutoff of their plates. The horizontal bars represent the mean of reactivity indexes for each group of patients studied. IND, indeterminate form; CARD, cardiac form; DIG, digestive form; CD, cardiodigestive form; FRA, flagellar repetitive antigen; OD, optical density; CO, cutoff.
DIG and CD forms [15]. In the present work we continue to study the humoral immune response in chronic Chagas disease, evaluating IgM serum levels against the CRA and FRA recombinant antigens of T. cruzi. We reported no significant differences between the levels of IgM for both recombinant antigens and the clinical forms in the chronic chagasic patients studied. SÂ Ferreira et al. [8] have observed no changes in IgM levels in any of the chronic clinical forms of T. cruzi infection. Matsumoto et al. [17], using immunofluorescence tests against amastigote and epimastigote crude antigens of T. cruzi, also reported no statistical differences between the presence of IgM antibodies and the chronic clinical forms of Chagas disease. However, these findings disagree with that of Morgan et al. [7], who observed high levels of IgM against epimastigote antigens of T. cruzi in CARD patients when compared with IND patients by ELISA. Although we observed that the patients with CARD and CD forms have slightly higher levels of IgM antibodies against the FRA antigen than that seen in the IND and DIG forms, these differences were not significant. These results do not support the hypothesis of Morgan et al. [7] that the presence of IgM against parasite antigens in the chronic phase of Chagas disease is strongly associated with autoimmune phenomena because we do not evaluate the crossreactivity of this class of antibodies with host antigens. Although we have found lower levels of IgM antibodies against CRA and FRA recombinant antigens of T. cruzi, a small proportion of the chronic chagasic patients analyzed in this study was positive for this isotype (CRA ⫽ 10.42%; FRA ⫽ 11.46%). Some authors who used ELISA to evaluate IgM antibodies also reported a lower numbers of patients with chronic T. cruzi infection who are positive for this class of antibody. BetÕnico et al. [10] demonstrated that 12.9% of chronic chagasic patients were positive for IgM using a synthetic tripeptide that consisted of a peptide containing 3 specific epitopes of trypomastigotes. Using cruzipain from epimastigotes as antigen, ZÛÒiga et al. [18] demonstrated 9.5% positivity among chronic chagasic patients, whereas Umezawa et al. [19], using trypomastigote excreted-secreted antigens, demonstrated only 3.0% positivity between chronic chagasic individuals. In addition to our study, these cited data [17–19] indicated lower positivity to anti-T. cruzi antibodies of the IgM class in the
Acknowledgments This work was supported by grants from Biomanguinhos/ Fiocruz and Conselho Nacional de Desenvolvimento CientÎfico e TecnolÔgico (CNPq). We thank Mineo Nakazawa for his technical assistance. We are also grateful to George Diniz for providing the statistical analysis. Y.M. Gomes is a CNPq research fellow. R.H.T. Vasconcelos is a Fiocruz PhD student. E.A.N. Azevedo is a graduate student and received a scholarship from PIBIC/Fiocruz. References [1] World Health Organization. Control of Chagas disease. WHO Tech Rep Ser 2002;905:109. [2] Dutra WO, Rocha MOC, Teixeira MM. The clinical immunology of human Chagas disease. Trends Parasitol 2005;21:581–7. [3] CorrËa-Oliveira R, Gomes JAS, Lemos EM, Cardoso GM, Reis DD, Adad S, et al. The role of the immune response on the development of severe clinical forms of human Chagas disease. Mem Inst Oswaldo Cruz 1999;94:253–5. [4] Lorca M, Gonzalez A, Veloso C, Reyes V, Vergara U. Immunodetection of antibodies in sera from symptomatic and asymptomatic Chilean Chagas’ disease patients with Trypanosoma cruzi recombinant antigens. Am J Trop Med Hyg 1992;46:44 –9. [5] Gazzinelli RT, Leme VMC, Cancado JR, Gazzinelli G, Scharfstein J. Identification and partial characterization of Trypanosoma cruzi antigens recognized by T cells and immune sera from patients with Chagas’ disease. Infect Immun 1990;58:1437– 44. [6] Magnani MAC, Ferriolli Filho F, de Siqueira AF. [Specific immunoglobulins (IgA, IgG, and IgM) in serum of patients with chronic Chagas’ disease analyzed by indirect immunofluorescence reactions]. Rev Inst Med Trop Sao Paulo 1973; 15:72–5. [7] Morgan J, Dias JCP, Gontijo ED, Bahia-Oliveira L, Correa-Oliveira R, Colley DG, et al. Anti-Trypanosoma cruzi antibody isotype profiles in patients with different clinical manifestations of Chagas’ disease. Am J Trop Med Hyg 1996;55:355–9. [8] SÂ Ferreira JA, GalvÄo-Castro B, Macedo W, Castro C. Immunoglobulins and other serological parameters in Chagas’ disease: evidence for increased IgA levels in the chronic digestive form. Clin Exp Immunol 1983;52:266 –70. [9] Primavera KSC, Umezawa ES, Peres BA, Camargo ME, Hoshino-Shimizu S. Chagas’ disease: IgA, IgM and IgG antibodies to T cruzi amastigote, trypomastigote and epimastigote antigens in acute and in different chronic forms of the disease. Rev Inst Med Trop Sao Paulo 1990;32:172– 80. [10] Umezawa ES, Shikanai-Yasuda MA, Stolf AMS. Changes in isotype composition and antigen recognition of anti-Trypanosoma cruzi antibodies from acute to chronic Chagas disease. J Clin Lab Anal 1996;10:407–13. [11] BetÕnico GN, Miranda EO, Silva DAO, Houghton R, Reed SG, Campos-Neto A, et al. Evaluation of a synthetic tripeptide as antigen for detection of IgM and IgG antibodies to Trypanosoma cruzi in serum samples from patients with Chagas disease or viral diseases. Trans R Soc Trop Med Hyg 1999;93:603– 6. [12] Lafaille JJ, Linss J, Krieger MA, Souto-PadrÔn T, de Souza W, Goldenberg S, et al. Structure and expression of two Trypanosoma cruzi genes encoding antigenic proteins bearing repetitive epitopes. Mol Biochem Parasitol 1989;35:127–36. [13] Krieger MA, Almeida E, Oelemann W, Lafaille JJ, Pereira JB, Krieger H, et al. Use of recombinant antigens for the accurate immunodiagnosis of Chagas’ disease. Am J Trop Med Hyg 1992;46:427–34. [14] VerÈosa AFA, Lorena VMB, Carvalho CL, Melo MFAD, Cavalcanti MGA, Silva ED, et al. Chagas’ disease: IgG isotypes against cytoplasmic (CRA) and flagellar (FRA) recombinant repetitive antigens of Trypanosoma cruzi in chronic chagasic patients. J Clin Lab Anal 2007;21:271– 6.
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[15] Vasconcelos RHT, Amaral FN, Cavalcanti MGAM, Silva ED, Ferreira AGP, Morais CNL, et al. Increased levels of IgA antibodies against CRA and FRA recombinant antigens of Trypanosoma cruzi differentiate digestive forms of Chagas disease. Hum Immunol 2010;71:964 –7. [16] Antas PRZ, Azevedo EM, Luz MRMP, Medrano-Mercado N, Chaves ACL, Vidigal PG, et al. A reliable and specific enzyme-linked immunosorbent assay for the capture of IgM from human chagasic sera using fixed epimastigotes of Trypanosoma cruzi. Parasitol Res 2000;86:813–20. [17] Matsumoto TK, Hoshino-Shimizu S, Nakamura PM, Andrade HF Jr, Umezawa ES. High resolution of Trypanosoma cruzi amastigote antigen in
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serodiagnosis of different clinical forms of Chagas’ disease. J Clin Microbiol 1993;31:1486 –92. [18] ZÛÒiga E, Montes C, Barbieri G, Gruppi A. Antibodies against Trypanosoma cruzi alkaline antigens are elicited in sera from acute but not chronic human chagasic patients. Clin Immunol 1999;93:81–9. [19] Umezawa ES, Nascimento MS, Stolf AMS. Enzyme-linked immunosorbent assay with Trypanosoma cruzi excreted-secreted antigens (TESA-ELISA) for serodiagnosis of acute and chronic Chagas’ disease. Diagn Microbiol Infect Dis 2001;39:169 –76.
Contents lists available at ScienceDirect
Immunoglobulin M antibodies against CRA and FRA recombinant antigens of Trypanosoma cruzi in chronic chagasic patients Romero H.T. Vasconcelos a, Elisa A.N. Azevedo a, Maria G.A.M. Cavalcanti b, Edimilson D. Silva c,d, Antonio G.P. Ferreira c,d, Clarice N.L. Morais a, Yara M. Gomes a,d,* a
Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhâes/Fiocruz, Recife, Brazil Hospital Universitàrio Oswaldo Cruz, Universidade de Pernambuco, Recife, Brazil c Departamento de Reativos para Diagnòstico, Bio-Manguinhos/Fiocruz, Rio de Janeiro, Brazil d Programa Integrado de Doenåa de Chagas/Fiocruz, Rio de Janeiro, Brazil b
A R T I C L E
I N F O
Article history: Received 29 October 2010 Accepted 22 February 2011 Available online 1 March 2011
Keywords: Immunoglobulin M Recombinant antigens Trypanosoma cruzi Chagas disease
A B S T R A C T
Previous works of our research group have demonstrated aspects of the humoral immune response of chronic Chagas disease using the cytoplasmatic repetitive antigen (CRA) and the flagellar repetitive antigen (FRA) of Trypanosoma cruzi. The aim of this work was to analyze the presence of specific immunoglobulin M (IgM) antibodies in chronic chagasic patients using these recombinant antigens of T. cruzi. The positivity of IgM in chronic chagasic patients against CRA and FRA antigens was determined by indirect enzyme-linked immunosorbent assay. We reported no statistical significant differences between the levels of IgM for both recombinant antigens and the different chronic clinical forms of Chagas disease. However, a small proportion of chronic chagasic patients analyzed in this study was positive for this antibody isotype. The findings of this study indicate that the IgM antibodies cannot be used to elucidate the differences in the profile of humoral immune response among chronic chagasic patients with different clinical forms using the CRA and FRA recombinant antigens of T. cruzi. 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction Chagas disease is a parasitic infection caused by the protozoan Trypanosoma cruzi. In the acute phase of the disease, usually developed with nonspecific symptoms, the blood parasitemia and the presence of immunoglobulin M (IgM) antibodies against T. cruzi are diagnostic criteria to confirm the infection [1]. Acute Chagas disease is followed by a chronic asymptomatic phase, also called indeterminate (IND) form. In this chronic form the patient enters a period of latency with no evidence of infection, other than positive serology for IgG against T. cruzi. About 20 to 30% of chronically infected individuals may develop severe forms with cardiac (CARD) and digestive (DIG) manifestations or both (CD) after 10 to 20 years of the initial infection [2]. The pathology of the lesions that lead to severe forms remains unknown [2,3]. However, some authors recognized that the human immune response to parasite antigens is related to the different clinical forms of the disease [3–5]. Magnani et al. [6] demonstrated that chronic chagasic patients have increased levels of IgM, probably because of antigenic variability of T. cruzi. Morgan et al. [7] demonstrated that chronic symptomatic patients present high levels of IgM against T. cruzi. According to Morgan et al. [7], this class of
* Corresponding author. E-mail address: yara@cpqam.fiocruz.br (Y.M. Gomes).
antibodies is first produced against T. cruzi antigens, but maintenance of this isotype in tissues and serum is directly associated with cross-reactivity among T. cruzi and host antigens. Although some studies have demonstrated the presence of IgM antibodies in the chronic phase of Chagas disease, only a few were conducted using pure and chemically defined antigens [8 –10]. BetÕnico et al. [11] compared the IgM positivity in chronic Chagas disease of a synthetic tripeptide with an alkaline extract of T. cruzi. The synthetic peptide exhibited a significantly lower number of positive samples when compared with the alkaline extract. In the present study we used two recombinant antigens of T. cruzi, chemically characterized and with a repetitive epitope structure, to evaluate the levels of IgM in chronic chagasic patients with different clinical forms. The cytoplasmatic repetitive antigen (CRA) has a repeated sequence of 14 amino acids and is expressed in epimastigote and amastigote forms of T. cruzi, whereas the flagellar repetitive antigen (FRA), present in the epimastigote and trypomastigote forms, has a repeated sequence of 68 amino acids [12,13]. VerÈosa et al. [14] studied the IgG isotypes against the CRA and FRA antigens in chronic chagasic patients and demonstrated that the IgG2 isotype against the FRA antigen was able to distinguish patients with the CARD form from those with the IND form. Recently, Vasconcelos et al. [15] studied the serum levels of IgA antibodies against these recombinant antigens and demonstrated that
0198-8859/11/$32.00 - see front matter 䉷 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2011.02.015
R.H.T. Vasconcelos et al. / Human Immunology 72 (2011) 402– 405
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the digestive forms of the disease present high levels of this isotype of immunoglobulin. Given the importance of the human immune response against the parasite antigens in the development and maintenance of chronic clinical forms of Chagas disease, the aim of the present study was to evaluate the levels of specific IgM against the CRA and FRA recombinant antigens of T. cruzi in the serum of chronic chagasic patients. It is possible that this isotype is involved in the pathology of severe clinical forms of the disease. 2. Subjects and methods 2.1. Study population Serum samples were collected from 96 chronic chagasic patients selected at the Oswaldo Cruz University Hospital of the State University of Pernambuco, Brazil. The selection of individuals was based on the following criteria: (1) clinical tests for characterization of the clinical forms as established by the World Health Organization [1]; (2) two positive serologic tests for Chagas disease; and (3) not having undergone etiologic treatment. According to their clinical status, the patients were classified as having IND (n ⫽ 24), CARD (n ⫽ 25), DIG (n ⫽ 20), or CD (n ⫽ 27) forms of the disease. To establish a cutoff point, serum was also collected from 14 non-chagasic individuals. Negative serologic results for infection by T. cruzi in two tests and never having received a blood transfusion were the inclusion criteria for this group.
Fig. 1. Reactivity indexes of immunoglobulin M against CRA antigen in chronic chagasic patients. Symbols represent the reactivity indexes, calculated as the mean OD of each sample divided by the cutoff of their plates. The horizontal bars represent the mean of reactivity indexes for each group of patients studied. IND, indeterminate form; CARD, cardiac form; DIG, digestive form; CD, cardiodigestive form; CRA, cytoplasmatic repetitive antigen; OD, optical density; CO, cutoff.
2.2. Ethical considerations All individuals involved in this study participated on a voluntary basis and signed the Terms of Free and Informed Consent. The methods of inclusion and the protocols used were approved by the Ethics Committee of the Aggeu MagalhÄes Research Center (Number 070/2008). 2.3. Recombinant antigens The CRA and FRA antigens of T. cruzi were prepared at the Bio-Manguinhos Department of Diagnostic Reactions and were obtained as previously described by Krieger et al. [13]. 2.4. Detection of IgM antibodies against CRA or FRA antigens Enzyme-linked immunosorbent assay (ELISA) plates (Nalge Nunc International Corporation, Rochester, NY) were coated with 100 L/well of CRA or FRA recombinant antigens at previously established concentrations (0.625 g/mL) diluted in a 0.05 mol/L carbonate– bicarbonate buffer, pH 9.6. After being blocked with phosphate-buffered saline containing 0.05% Tween 20 (PBST) and 1% bovine serum albumin, the plates were incubated with serum diluted 1:100 in PBST and then with biotin-conjugated immunoglobulin (anti-IgM; Sigma, St. Louis, MO) diluted 1:4000 in PBST and 1% bovine serum albumin and subsequently with peroxidaseconjugated streptavidin (GE Healthcare, Buckinghamshire, UK) diluted 1:6000 in PBST. The immunocomplex was revealed by the addition of 0.01% o-phenylenediamine and 0.01% hydrogen peroxide diluted in a 0.077 mol/L citrate–phosphate buffer, pH 5.0. The reaction was terminated by adding 2.5 N sulfuric acid, and the absorbance was measured using an ELISA plate reader (Bio-Rad 3550, Bio-Rad Laboratories, Vienna, VA) at 490 nm. All samples were tested in duplicate. The cutoff point was established for each plate as the mean of optical density (OD) of non-chagasic individuals plus 2-fold standard deviation and the results were expressed as a reactivity index, as previously described by Antas et al. [16], whereby the value of the mean of the OD of each sample was divided by the cutoff value of their respective plates to enable comparison of the results from different plates. Reactivity indexes with values higher than 1.0 were considered positive [16].
2.5. Statistical analysis The R software package was used to analyze the results. Bartlett, analysis of variance, and Tukey tests were applied to evaluate the differences between the indexes calculated for the samples in each group of patients. Data are presented as means ⫾ standard error of the mean, and the significance level for all conclusions was set at 5%. 3. Results The mean levels of IgM antibodies against CRA or FRA recombinant antigens of T. cruzi were just low in almost all chronic chagasic patients studied. However, a small proportion of chronic chagasic patients had high levels of this isotype. The positivity of IgM antibodies in the serum of the chronic chagasic patients evaluated in this study was 10.42% for the CRA antigen and 11.46% for the FRA antigen. No significant statistical differences were observed in the serum levels of IgM when we compared the patients with different clinical forms by their reactivity indexes using the CRA antigen (Fig. 1). The FRA antigen antibody isotype was higher in patients with the CARD and CD forms when compared with patients with the IND and DIG forms, although these differences were not statistically significant (Fig. 2). 4. Discussion The immunologic basis for the variation of clinical manifestations among patients with chronic T. cruzi infection remains unclear [2]. Previous studies recognized that a particular antibody isotype may be related to chronic clinical manifestations of the disease [4 – 8]. This observation led us to examine a possible association between antibody isotype profile against CRA and FRA recombinant antigens of T. cruzi in chronic chagasic patients and different chronic clinical forms of the disease, with a view to using these antibodies as prognostic markers for the evolution of Chagas disease. In previous works of our research group, the IgG2 isotype against the FRA antigen was associated with the CARD form [14] and the IgA isotype against both antigens was associated with the
404
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chronic phase of Chagas disease. The differences observed in the percentage of positive samples likely occurred because the antigens used in the studies do not share the same epitopes. These findings agree with the hypothesis that anti-T. cruzi IgM antibodies are only occasionally reported in chronic chagasic patients because of antigenic variability of the parasite [10]. According to Magnani et al. [6], the constant antigenic variability is a strategy of immune evasion used by T. cruzi and other protozoans to survive and reproduce inside the host. Moreover, the hypothesis that this isotype of antibodies are produced and maintained in the chronic phase of Chagas disease because of the cross-reactivity with host antigens [7] is not supported by these studies. If this hypothesis were correct, almost all chronic chagasic patients would present high levels of IgM because of producing an immune response against selfantigens. Taking the results of this study into account, the IgM antibodies against the CRA and FRA recombinant antigens of T. cruzi cannot be used to elucidate differences in the profile of humoral immune response among chronic chagasic patients with different clinical forms. Fig. 2. Reactivity indexes of IgM against FRA antigen in chronic chagasic patients. Symbols represent the reactivity indexes, calculated as the mean OD of each sample divided by the cutoff of their plates. The horizontal bars represent the mean of reactivity indexes for each group of patients studied. IND, indeterminate form; CARD, cardiac form; DIG, digestive form; CD, cardiodigestive form; FRA, flagellar repetitive antigen; OD, optical density; CO, cutoff.
DIG and CD forms [15]. In the present work we continue to study the humoral immune response in chronic Chagas disease, evaluating IgM serum levels against the CRA and FRA recombinant antigens of T. cruzi. We reported no significant differences between the levels of IgM for both recombinant antigens and the clinical forms in the chronic chagasic patients studied. SÂ Ferreira et al. [8] have observed no changes in IgM levels in any of the chronic clinical forms of T. cruzi infection. Matsumoto et al. [17], using immunofluorescence tests against amastigote and epimastigote crude antigens of T. cruzi, also reported no statistical differences between the presence of IgM antibodies and the chronic clinical forms of Chagas disease. However, these findings disagree with that of Morgan et al. [7], who observed high levels of IgM against epimastigote antigens of T. cruzi in CARD patients when compared with IND patients by ELISA. Although we observed that the patients with CARD and CD forms have slightly higher levels of IgM antibodies against the FRA antigen than that seen in the IND and DIG forms, these differences were not significant. These results do not support the hypothesis of Morgan et al. [7] that the presence of IgM against parasite antigens in the chronic phase of Chagas disease is strongly associated with autoimmune phenomena because we do not evaluate the crossreactivity of this class of antibodies with host antigens. Although we have found lower levels of IgM antibodies against CRA and FRA recombinant antigens of T. cruzi, a small proportion of the chronic chagasic patients analyzed in this study was positive for this isotype (CRA ⫽ 10.42%; FRA ⫽ 11.46%). Some authors who used ELISA to evaluate IgM antibodies also reported a lower numbers of patients with chronic T. cruzi infection who are positive for this class of antibody. BetÕnico et al. [10] demonstrated that 12.9% of chronic chagasic patients were positive for IgM using a synthetic tripeptide that consisted of a peptide containing 3 specific epitopes of trypomastigotes. Using cruzipain from epimastigotes as antigen, ZÛÒiga et al. [18] demonstrated 9.5% positivity among chronic chagasic patients, whereas Umezawa et al. [19], using trypomastigote excreted-secreted antigens, demonstrated only 3.0% positivity between chronic chagasic individuals. In addition to our study, these cited data [17–19] indicated lower positivity to anti-T. cruzi antibodies of the IgM class in the
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